A Rapid Real Time Fluorescence Recombinase-aided Amplification Method for Detecting Porcine Pseudorabies Virus
[Objective]A rapid method applying the recombinase-aided amplification(RAA)method for detecting porcine pseudorabies virus(PRV)was developed.[Methods]Based on the sequence of gE gene in PRV,specific primers and probes were designed.Amplification conditions were optimized,and assay specificity,sensitivity,and reproducibility scrutinized by a verification trial on clinical samples.[Results]The newly developed assay successfully amplified the PRV nucleic acids in merely 23m under the constant temperature of 43℃with a detection limit of 111 copies·μL-1.There were no cross reactions with viruses that produced reproductive and respiratory syndromes,epidemic diarrhea,rotavirus,transmissible gastroenteritis,circovirus 2,or circovirus 3 on pigs.The coefficients of variation within a group and between groups on the repeatability test were less than 5%.And,on 40 clinical samples,the positive detection on PRV of the assay was 15%(6/40),which was comparable to that of the conventional PCR.[Conclusion]A simple,rapid,efficient,and accurate method of fluorescence RAA detection on PRV was established for laboratory testing and epidemiological investigation of the disease.
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